Colorectal carcinoma peritoneal metastases-derived organoids: results and perspective of a model for tailoring hyperthermic intraperitoneal chemotherapy from bench-to-bedside

Background Peritoneal metastases from colorectal cancer (CRCPM) are related to poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been reported to improve survival, but peritoneal recurrence rates are still high and there is no consensus on the drug of choice for HIPEC. The aim of this study was to use patient derived organoids (PDO) to build a relevant CRCPM model to improve HIPEC efficacy in a comprehensive bench-to-bedside strategy. Methods Oxaliplatin (L-OHP), cisplatin (CDDP), mitomycin-c (MMC) and doxorubicin (DOX) were used to mimic HIPEC on twelve PDO lines derived from twelve CRCPM patients, using clinically relevant concentrations. After chemotherapeutic interventions, cell viability was assessed with a luminescent assay, and the obtained dose–response curves were used to determine the half-maximal inhibitory concentrations. Also, induction of apoptosis by different HIPEC interventions on PDOs was studied by evaluating CASPASE3 cleavage. Results Response to drug treatments varied considerably among PDOs. The two schemes with better response at clinically relevant concentrations included MMC alone or combined with CDDP. L-OHP showed relative efficacy only when administered at low concentrations over a long perfusion period. PDOs showed that the short course/high dose L-OHP scheme did not appear to be an effective choice for HIPEC in CRCPM. HIPEC administered under hyperthermia conditions enhanced the effect of chemotherapy drugs against cancer cells, affecting PDO viability and apoptosis. Finally, PDO co-cultured with cancer-associated fibroblast impacted HIPEC treatments by increasing PDO viability and reducing CASPASES activity. Conclusions Our study suggests that PDOs could be a reliable in vitro model to evaluate HIPEC schemes at individual-patient level and to develop more effective treatment strategies for CRCPM. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03052-5.

Immunohistochemistry analyses FFPE blocks were prepared for immunohistochemistry (IHC) as previously described [1].PDOs were included in paraffin and cut into slices (3 µm); paraffin was removed with xylene and slices were rehydrated using decreasing concentrations of ethanol, and washed with water.IHC was performed with the following antibodies: CK AE1/AE3, CK20, CDX2 and ki-67, following the specifications reported in Supplementary Table S6.Images were acquired with a DM6000B microscope (Leica).Antigen retrieval was carried out using preheated target retrieval solution (pH 6.0) for 30 minutes.Tissue sections were blocked with FBS serum in PBS for 60 min and incubated overnight with primary antibody.The antibody binding was detected using a polymer detection kit (GAM/GAR-HRP, Microtech, Italy) followed by a diaminobenzidine chromogen reaction (Peroxidase substrate kit, DAB, SK-4100; Vector Lab, USA).All sections were counterstained with Mayer's hematoxylin.

QuPath analyses
Cell counting and percentage estimation of cCASPASE3-positive cells were performed using Qupath software (https://qupath.github.io,version 0.2.3).The percentage of positive cells was calculated by dividing the number of positive cells present in each field by the total number of cells (cCASPASE + + cCASPASE3 -) in the same field.

CellTiterGlo® 3D Cell Viability Assay and Caspase-Glo® 3 Assay protocol
PDOs and co-culture viability and CASPASE 3 activation were assessed using a CellTiterGlo® 3D Cell Viability Assay kit and a Caspase-Glo® 3 assay, respectively (Promega, USA) on a TECAN spark microplate reader (Tecan Trading AG, Switzerland) following the manufacturers' instructions.Data were normalized to the mean of the untreated group (UNT: PDO treated with DMEM-F12 only).All the experiments were performed in triplicate.

Immunoblotting: Cancer -associated fibroblast characterization
CAF were lysed and their protein content was extracted as in [1].For each sample, 40 μg of protein extract were separated on 4-12% polyacrylamide gels and incubated with the following primary antibodies: α-SMA; FAP; E-Cadherin and Vinculin.The signals were detected using enhanced chemiluminescence, and protein levels were quantified using Image Lab Software (Bio-Rad, Hercules, CA, USA) (Supplementary Table S3).

Table S1 :
The complete list of growth factors and media supplements, with their working concentrations used for TDOs culturing.